Abstract
We implement wave front sensor-less adaptive optics in a structured illumination microscope. We investigate how the image formation process in this type of microscope is affected by aberrations. It is found that aberrations can be classified into two groups, those that affect imaging of the illumination pattern and those that have no influence on this pattern. We derive a set of aberration modes ideally suited to this application and use these modes as the basis for an efficient aberration correction scheme. Each mode is corrected independently through the sequential optimisation of an image quality metric. Aberration corrected imaging is demonstrated using fixed fluorescent specimens. Images are further improved using differential aberration imaging for reduction of background fluorescence.
Highlights
Optical sectioning microscopy is widely used to provide three-dimensional fluorescence images of biological specimens
We demonstrated the aberration correction scheme in a structured illumination microscope based around a modified IX70 inverted microscope (Olympus), incorporating a deformable
The grid was illuminated by a white-light source with a narrowband excitation filter centered on 488 nm (Chroma) and imaged in the focal plane of the objective after being reflected off the deformable mirror (DM), which was conjugated to the pupil plane of the objective
Summary
Optical sectioning microscopy is widely used to provide three-dimensional fluorescence images of biological specimens. An alternative is to use a wide-field technique such as structured illumination (SI) microscopy, which retains the sectioning ability of confocal microscopy, but can be implemented in a conventional microscope using an incoherent light source, and without the need for scanning [3] In this technique, the image of a grid is projected on the specimen so as to produce a one-dimensional sinusoidal excitation pattern in the focal plane of the objective lens. By using an appropriate combination of optimisation metric, modal aberration expansion and aberration estimator algorithm, correction can be achieved with a minimal number of measurements The use of these efficient correction schemes is of particular interest in biological imaging, where the reduced number of measurements minimises photobleaching and damage on the sample. We discuss how the general scheme presented in this paper can be applied to other adaptive optics systems
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